Page 301 - The Veterinary Laboratory and Field Manual 3rd Edition

270 Susan C. Cork and Roy Halliwell

Figure 4.32 (a) Thermocycler used in conventional (a)
PCR technique. This equipment provides a cycling
temperature for amplification of nucleic acid and the
end product detection is done by gel electropho-
resis. (b) Loading of PCR products onto a agarose
gel for electrophoresis. (c) Gel electrophoresis of
conventional PCR products. Template was ampli-
fied with three pairs of porcine circovirus capsid
gene specific primers and the samples were run in
a 2% agarose gel. Lane 1: DNA ladder; lanes 2–4
pig number 1 three different primers, lane 5–7 pig
number 2 three different primers, lane 8 positive (b)
control only one primer and lane 9 no template
control. Photos: (a) Dr Davor Ojkic, Animal Health
Laboratory, University of Guelph, Canada; (b) Dr
Markus Czub, University of Calgary, Canada.

therefore, can be used for RNA viruses too. The
change in fluorescent signal as double stranded
DNA dissociates with increased temperature,
may also be coupled with real-time technology in (c)
a process called high resolution melting analysis
(HRM). This is very sensitive and can be utilized
for analysis of SNPs in the targeted sequences.
Real-time PCR therefore has applications in
detecting small quantities of DNA or RNA in
specimen samples, can be used for identification
of biomarkers such as antibiotic resistance genes
or SNPs for strain identification or epidemiologi-
cal studies and, because of its good quantitative
capabilities, be used to monitor disease progress
or treatment, for example, tuberculosis. SYBR green-based, real-time PCR assay does not
In virology, real-time PCR methods based require probes and, therefore, is economical and
on different detection systems have been devel- easily adoptable from the conventional PCR sys-
oped for the quantification of the viral genome tem. Further, the SYBR green-based, real-time
in different tissues. These methods are depen- PCR assay detects the target PCR product accu-
dent on TaqMan/hybridization probes or using mulation independent of the sequence; as such,
SYBR green chemistry and determine the viral it allows the quantification of the viral genome
genome loads based on either relative increase with minor variations in the sequence. This may
of viral genomes and gene transcription levels, not be possible with probes as they operate in
but not virion replication in vitro, or absolute a highly target sequence-specific manner and
quantification. Both real-time PCR methods small changes in target sequence may abrogate
are equally efficient in quantifying viruses. The probe binding leading to the generation of false

Vet Lab.indb 270 26/03/2019 10:25